Microorganism for producing purine nucleotide and method for producing purine nucleotide using same

Abstract

The present disclosure relates to a microorganism whose formate dehydrogenase activity is weakened compared to its endogenous activity; a method of producing purine nucleotides comprising: culturing a microorganism in a culture medium; a composition for producing purine nucleotides, comprising a microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more of the above; and the use of a microorganism for producing purine nucleotides.

Description

[Technical field]

[0001] The present disclosure relates to a microorganism whose formate dehydrogenase activity is weakened compared with its endogenous activity; a method for producing purine nucleotides, comprising: culturing the microorganism in a culture medium; a composition for producing purine nucleotides, comprising the microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more of the above; and use of the microorganism for the production of purine nucleotides. [Background technology]

[0002] Purine nucleotides (i.e., 5'-inosine monophosphate (IMP), 5'-xanthine monophosphate (XMP), and 5'-guanosine monophosphate (GMP)) are intermediates in the nucleic acid biosynthesis metabolic system, have important physiological functions in the body, and are widely used in food, medicine, etc. Specifically, inosinic acid is considered to have a beefy flavor, while GMP derived from XMP is considered to have a mushroom flavor. Both materials are believed to enhance the flavor intensity of monosodium glutamate (MSG), and therefore have attracted attention as a flavor-rich nucleic acid-based condiment.

[0003] Meanwhile, examples of a method for producing purine nucleotides include: (1) a method for degrading ribonucleic acid (RNA) extracted from yeast cells with an enzyme; (2) a fermentation preparation method for directly recovering purine nucleotides in a culture solution by culturing a microorganism that produces purine nucleotides; (3) a method for chemically phosphorylating nucleosides produced by fermentation; (4) a method for enzymatic phosphorylation of nucleosides produced by fermentation, etc. (Korean Patent Publication No. 10-1049023, Japanese Patent Publication No. 4363042, Korean Patent Publication No. 10-1210704 and Agri. Biol. Chem., 36 (9), 1511-1522). Among them, method (1) has problems in terms of raw material supply and demand and economic benefits, while method (2) is widely used due to its economic and environmental advantages. Meanwhile, in the case of GMP production of one of purine nucleotides, due to its cell membrane permeability, there is a disadvantage of low yield, so a production method of enzymatic conversion of XMP produced by microbial fermentation into GMP is also used.

[0004] However, in the process of producing purine nucleotides by using microbial fermentation, the microorganisms may be stressed due to temperature, pH value, osmotic pressure, malnutrition and oxidative factors. Among them, especially in oxidative stress, reactive oxygen species (ROS) as an inevitable factor produced in the fermentation production process, become the main cause, and ROS may cause abnormal growth of microorganisms.

[0005] Therefore, there is still a need to explore efficient purine nucleotide production. [Summary of the invention]

[0006]

Technical issues

[0007] The present inventors have developed a microorganism whose formate dehydrogenase activity is weakened compared with its endogenous activity; a method for producing purine nucleotides, which comprises: culturing the microorganism in a culture medium; a composition for producing purine nucleotides, which comprises the microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more of the above; and use of the microorganism for producing purine nucleotides, thereby completing the present invention.

[0008] [Technical solution to the problem]

[0009] One of the objects of the present disclosure is to provide a microorganism having a purine nucleotide-producing ability, whose formate dehydrogenase activity is weakened compared with its endogenous activity.

[0010] In one embodiment, the formate dehydrogenase may consist of any one or more amino acid sequences selected from SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and combinations thereof.

[0011] In the microorganism according to any of the above embodiments, the activity of any one or more proteins selected from the following (a) to (c) may be further regulated:

[0012] (a) the activity of glycine degrading enzyme is enhanced compared with its endogenous activity;

[0013] (b) the serine hydroxymethyltransferase activity is enhanced compared to its endogenous activity; and

[0014] (c) Formate-dependent phosphoribosylglycinamide formyltransferase activity is enhanced compared to its endogenous activity.

[0015] In the microorganism of any of the above embodiments, the microorganism may be a microorganism of the genus Corynebacterium.

[0016] In the microorganism of any of the above embodiments, the Corynebacterium microorganism may be Corynebacterium stationis.

[0017] In the microorganism of any of the above embodiments, the microorganism may have enhanced purine nucleotide production ability compared to an unmodified microorganism.

[0018] Another object of the present disclosure is to provide a method for producing purine nucleotides, comprising: culturing a microorganism in a culture medium, wherein the activity of formate dehydrogenase of the microorganism is attenuated relative to its endogenous activity.

[0019] In one embodiment, the method further comprises recovering the target substance from the cultured microorganism, the culture product of the microorganism, the fermentation product of the microorganism or the culture medium.

[0020] Another object of the present application is to provide a composition for producing purine nucleotides, which comprises: a microorganism in which the activity of formate dehydrogenase is weakened relative to its endogenous activity, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more of the above.

[0021] Another object of the present application is to provide a use of a microorganism for producing purine nucleotides, wherein the formate dehydrogenase activity of the microorganism is weakened compared to its endogenous activity.

[0022]

Beneficial effects

[0023] The microorganism having attenuated formate dehydrogenase activity compared to its endogenous activity disclosed herein can produce purine nucleotides at a high yield, and thus can be effectively used in the industrial production of purine nucleotides.

[0024] [Implementation Method]

[0025] The content of the present invention is described in detail below. At the same time, each description and embodiment disclosed herein can be applied to other descriptions and embodiments respectively. In other words, all combinations of various elements disclosed herein belong to the scope of the present invention. In addition, the scope of the present invention is not limited by the specific description described below.

[0026] Furthermore, those skilled in the art may identify or ascertain, using no more than routine experimentation, numerous equivalents to the specific aspects of the invention described herein. Additionally, such equivalents are intended to be encompassed by the present invention.

[0027] As used in the specification and the appended claims, the singular forms ("a," "an," and "the") include plural referents unless the context clearly dictates otherwise. Singular terms shall include pluralities and plural terms shall include the singular unless the context dictates otherwise. As used in the specification and the appended claims, the use of "or" may be intended to include "and / or" unless otherwise indicated.

[0028] As used herein, the term "about" may be presented before a particular numerical value. The term "about" as used herein includes not only the exact number listed after the term, but also a range that is close to or near that number. Considering the context of the presented numbers, it can be determined whether there are any numbers that are close to or near the particular number presented. In one example, the term "about" may refer to a range between -10% and +10% of the numerical value. In another example, the term "about" may refer to a range of -5% to +5% of a given numerical value, but is not limited thereto.

[0029] As used herein, descriptions such as the terms "first, second, third...", "(i), (ii), (iii)...", or "(a), (b), (c), (d)..." are used to distinguish similar components, and these terms do not mean that the components are performed sequentially or in sequence. For example, when the terms are used to refer to steps of a method, use, or assay, there may be no time interval between these steps, or they may be performed simultaneously, or they may be performed a few seconds, minutes, hours, days, or months apart.

[0030] As used herein, the term "consisting essentially of may mean that an unspecified component may be present, provided that characteristics of the subject matter claimed herein are not substantially affected by the presence of the unspecified component.

[0031] The term "consisting of..." as used herein means that the total ratio of the specific components is 100%. The components or features listed after the term "consisting of" may be essential or mandatory. In some embodiments, in addition to the components or features listed after the term "consisting of...", any other components or non-essential components may be excluded.

[0032] The term "includes / comprises" as used herein refers to the presence of the features, steps or components described after the term, and does not exclude the presence or addition of one or more features, steps or components. The components or features listed after the term "includes / comprises" herein may be essential or mandatory. However, in some embodiments, the term may also include any other or non-essential components or features.

[0033] One aspect of the present disclosure provides a microorganism having a purine nucleotide-producing ability, wherein the formate dehydrogenase activity is attenuated compared with its endogenous activity.

[0034] Attenuation of formate dehydrogenase activity can be defined as: the purine nucleotide-producing ability of the microorganism of the present disclosure is increased compared to the production ability of a natural wild-type microorganism or a non-modified microorganism (e.g., a microorganism expressing a polypeptide having wild-type formate dehydrogenase activity (e.g., a polypeptide selected from SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and a combination thereof) or a strain in which the formate dehydrogenase activity of the present disclosure is not attenuated compared to the endogenous activity or a strain before attenuation), but is not limited thereto.

[0035] For example, the activity of formate dehydrogenase can be evaluated by measuring the productivity or yield of purine nucleotides, but is not limited thereto.

[0036] The term "formate dehydrogenase" as used herein is an enzyme composed of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3), which utilizes formate as a substrate and catalyzes an oxidation reaction to reduce NAD + And produce NADH and CO2. Specifically, the formate dehydrogenase of the present application can be any one or more proteins selected from formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3), but is not limited thereto. The formate dehydrogenase of the present application can be used to refer to the complex of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3), and can be used interchangeably with Fdh.

[0037] As used herein, "fdh gene", which consists of fdh1, fdh2 and fdh3 genes, can be used to refer to the gene encoding formate dehydrogenase in the present application. Specifically, the fdh gene of the present application can be used to refer to the gene encoding formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3) complex.

[0038] Specifically, the formate dehydrogenase of the present application can be a protein having the activity of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3) encoded by fdh1, fdh2 and fdh3 genes, respectively, and its type is not particularly limited as long as the protein has the activity corresponding to formate dehydrogenase. Formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3) encoded by fdh1, fdh2 and fdh3 genes, respectively, are known in the art, and the amino acid and polynucleotide sequences of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3) can be obtained from known databases, such as GenBank of NCBI, etc., but are not limited thereto.

[0039] In one example, formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3) may comprise the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, respectively, or amino acid sequences having at least 60% homology or identity thereto, but are not limited thereto, as long as it has formate dehydrogenase activity. Specifically, the polypeptides having formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3) activity may have or comprise the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, respectively, or amino acid sequences having at least 60%, 70%, 80%, 85%, 90%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or identity with SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, respectively, or consist of or essentially consist of. In one example, formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3) may refer to proteins endogenously present in a Corynebacterium microorganism or Corynebacterium stationis, but are not limited thereto. Specifically, formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3) may be formate dehydrogenase subunit 1 (Fdh subunit 1) composed of the amino acid sequence of SEQ ID NO: 39, formate dehydrogenase subunit 2 (Fdh subunit 2) composed of SEQ ID NO: 41, and formate dehydrogenase subunit 3 (Fdh subunit 3) composed of the amino acid sequence of SEQ ID NO: 43, which are endogenously present in a Corynebacterium microorganism or Corynebacterium stationis, but are not limited thereto.

[0040] Regarding the amino acid sequence of the present application, although it is described as a polypeptide "comprising / including" the amino acid sequence described by a specific sequence number, a polypeptide "composed of" the amino acid sequence described by a specific sequence number, or a polypeptide or protein "having" the amino acid sequence described by a specific sequence number, it is obvious that any protein with a partial sequence deletion, modification, substitution, conservative substitution or addition in the amino acid sequence can be used in the present application if it has the same or corresponding activity as the protein composed of the amino acid sequence of the corresponding sequence number. For example, sequence additions, naturally occurring mutations, silent mutations or conservative substitutions upstream or downstream of the amino acid sequence that do not change the function of the protein are not excluded, as long as the protein has the same or corresponding activity as the modified protein, it is obvious to those skilled in the art that such sequence additions or mutations fall within the scope of the present invention.

[0041] For example, it can be an addition, a spontaneous mutation, a silent mutation or a conservative substitution that does not change the mutant polypeptide function of the present application at the N-terminal, C-terminal and / or internal addition of the amino acid sequence. For example, the polypeptide can be coupled to an N-terminal signal (or leader) sequence that participates in co-translation or post-translational protein translocation. In addition, the polypeptide can also be coupled to another sequence or a connecting peptide to identify, purify or synthesize the polypeptide.

[0042] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Amino acids can be divided into the following groups: In one example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with non-polar side chains (non-polar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, amino acids with charged side chains include arginine, lysine, histidine, glutamic acid, aspartic acid; amino acids with uncharged amino acids (called neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine and glutamine. In another example, aromatic amino acids include phenylalanine, tryptophan and tyrosine. In another example, branched-chain amino acids include valine, leucine and isoleucine. In another example, 20 amino acids can be divided into 5 groups according to their size, starting with the relatively small amino acid group, namely glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan and tyrosine, but the classification of amino acids is not limited thereto. Generally, conservative substitutions may have little or no effect on the activity of a polypeptide.

[0043] Furthermore, the nucleotide sequence encoding formate dehydrogenase may be a nucleotide sequence encoding a protein exhibiting formate dehydrogenase activity.

[0044] For example, the nucleotide sequences encoding formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2), and formate dehydrogenase subunit 3 (Fdh subunit 3) may be nucleotide sequences encoding proteins exhibiting formate dehydrogenase activity.

[0045] For example, formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3) having the amino acid sequences of SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:43, respectively, may be encoded by a polynucleotide having or comprising the nucleotide sequences of SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44, or nucleotide sequences having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more and less than 100% homology or identity with SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44, or consisting of or consisting essentially of, but not limited to, these. In addition, the nucleotide sequences of SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44 can be obtained from known databases such as GenBank of NCBI, but are not limited thereto.

[0046] In the present disclosure, for example, a gene comprising the nucleotide sequence of SEQ ID NO: 40 may be used interchangeably with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 40, a gene or polynucleotide having the nucleotide sequence of SEQ ID NO: 40, and a gene or polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 40.

[0047] Taking into account the codon degeneracy or the preferred codons of the organism expressing the formate dehydrogenase of the present application, the polynucleotide of the present application can be variously modified in the coding region without changing the amino acid sequence of the formate dehydrogenase of the present application. Therefore, based on the codon degeneracy, it is obvious that a polynucleotide that can be translated into a polypeptide comprising the amino acid sequence of the formate dehydrogenase of the present application or a polynucleotide having homology or identity thereto can also be included. For example, the polynucleotide of the present disclosure can be SEQ ID NO: 40, SEQ ID NO: 42 and / or SEQ ID NO: 44, or a degenerate sequence thereof.

[0048] In another example, the polynucleotide of the present application may have or include a nucleotide sequence having more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO:40, SEQ ID NO:42 and / or SEQ ID NO:44, or consist of or essentially consist of a nucleotide sequence having more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO:40, SEQ ID NO:42 and / or SEQ ID NO:44, but is not limited thereto.

[0049] In addition, the polynucleotides of the present application may include probes that can be prepared from known gene sequences, for example, any polynucleotide sequence is not limited as long as it can hybridize with a sequence complementary to all or part of the polynucleotide sequence of the present application under stringent conditions and encode the formate dehydrogenase of the present application.

[0050] As used herein, the term "homology" or "identity" refers to the degree of relatedness between two given amino acid sequences or nucleotide sequences and can be expressed as a percentage. The two terms homology and identity are often used interchangeably.

[0051] The sequence homology or identity of conservative polynucleotides or polypeptides can be determined by standard alignment algorithms and can be used with the default gap penalty established by the program used. Substantially homologous or identical sequences will typically hybridize to at least about 50%, 60%, 70%, 80% or 90% of the full length of the sequence under moderate or high stringency conditions. Polynucleotides containing degenerate codons rather than codons in the hybridizing polynucleotides are also contemplated.

[0052] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by using a known computer algorithm such as the "FASTA" program using default parameters such as in Pearson et alia (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et alia, 2000, Trends Genet. 16: 276-277) (version 5.0 or higher) (GCG program package (Devereux, J. et alia, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et alia, J MOLEC BIOL 215: 403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et alia (1988) SIAM J Applied Mater. Math 48:1073)). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0053] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program mentioned in Needleman et alia (1970), J Mol Biol. 48: 443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In summary, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of the shorter symbols in the two sequences. Default parameters for the GAP program may include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity), a PAM matrix (refer to the content disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)), the weighted comparison matrix of Gribskov et alia (1986) Nucl Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 points for each gap and an additional penalty of 0.10 points for each symbol in each gap (or a penalty of 10 points for gap opening and a penalty of 0.5 points for gap extension); (3) no penalty for end gaps.

[0054] In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. The defined appropriate hybridization conditions are within the scope of the present technology and can be determined by methods known to those skilled in the art (e.g., J. Sambrook et alia, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY, 1989; FM Ausubel et alia, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0055] "Stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are disclosed in detail in the literature (e.g., J. Sambrook et alia, supra). For example, stringent conditions may include conditions that allow hybridization between polynucleotides with high homology or identity, between polynucleotides with more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% homology or identity, and conditions that do not hybridize between polynucleotides with homology or identity lower than the above homology or identity; or may include washing conditions for conventional Southern blot hybridization, i.e., washing once, particularly twice or three times, at a salt concentration and temperature equivalent to 60°C., 1×SSC, 0.1% SDS, particularly 60°C., 0.1×SSC, 0.1% SDS, and more particularly 68°C., 0.1×SSC, 0.1% SSD.

[0056] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that may hybridize with each other. For example, in the case of DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application not only include nucleotide sequences that are substantially similar thereto, but may also include separate nucleic acid fragments that are complementary to the full-length sequence.

[0057] For example, polynucleotides homologous or identical to the polynucleotides of the present application can be prepared using a T of 55°C. m The hybridization conditions of the step of hybridization are determined by using the above conditions. m The value may be 60° C., 63° C., or 65° C., but is not limited thereto, and may be appropriately adjusted by those skilled in the art according to their purpose.

[0058] The appropriate stringency for hybridization of polynucleotides depends on the length of the polynucleotides and the degree of complementation, and these variables are well known in the art (see Sambrook et alia, supra, 9.50-9.51, 11.7-11.8).

[0059] The term "purine nucleotide" used herein may specifically be any one or more nucleotides selected from 5'-inosine monophosphate (hereinafter referred to as IMP), 5'-xanthine monophosphate (hereinafter referred to as XMP) and 5'-guanosine monophosphate (hereinafter referred to as GMP). IMP is a deaminated adenine compound, which refers to a nucleotide composed of a single hypoxanthine, ribose and phosphate. IMP can be biosynthesized by 5'-phosphoribosyl-1-pyrophosphate (PRPP), specifically, it can be formed by replacing the pyrophosphate group bound to the first carbon of PRPP with a nitrogen atom and forming an imidazole ring and a pyrimidine ring through nine steps. XMP refers to a nucleotide produced by dehydrogenation of IMP, which can be synthesized from IMP by inosine-5'-monophosphate dehydrogenase. GMP refers to a nucleotide having a phosphate group forming an ester bond structure in the ribose of a guanine nucleotide molecule. GMP can be synthesized by adding an ammonia molecule to XMP by 5'-guanylate biosynthase (GMP synthase). The method for preparing GMP from XMP and / or the means used in the method can be selected from known technologies.

[0060] The term "microorganism (or strain)" used herein includes all wild-type microorganisms, or natural or artificial genetic modifications and microorganisms that weaken or enhance specific mechanisms due to the insertion of exogenous genes or the enhancement or inactivation of endogenous genes, and can be a microorganism including genetic modification to produce target polypeptides, proteins, and products. In this application, "microorganism" and "strain" have the same meaning and can be used interchangeably without restriction.

[0061] For example, the microorganism of the present disclosure may be a microorganism (eg, a recombinant strain) in which the activity of formate dehydrogenase is attenuated compared to endogenous activity, but is not limited thereto.

[0062] The term "microorganism with purine nucleotide production ability" as used herein refers to a prokaryotic or eukaryotic microbial strain that can produce purine nucleotides in an organism, and may include all of the following: microorganisms that impart purine nucleotide production ability to a parent strain that does not have purine nucleotide production ability or microorganisms that endogenously have purine nucleotide production ability. The production ability of purine nucleotides can be imparted or enhanced by species improvement.

[0063] The term "non-modified microorganism" as used herein does not exclude strains containing mutations that may occur naturally in microorganisms, and may refer to wild-type strains or natural strains themselves, or strains before their properties are changed due to genetic modification caused by natural or artificial factors. "Non-modified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "non-modified strain", "non-mutated microorganism", "parent strain before mutation", "wild-type microorganism", "reference microorganism" or "standard microorganism". In the present disclosure, non-modified microorganisms may refer to strains whose formate dehydrogenase activity of the present disclosure is not weakened compared to the endogenous activity of formate dehydrogenase, or strains before weakening, but are not limited to this. In addition, in the present disclosure, non-modified microorganisms may be microorganisms comprising an amino acid sequence consisting of SEQ ID NO: 39, SEQ ID NO: 41 and / or SEQ ID NO: 43, or microorganisms consisting of a polynucleotide consisting of SEQ ID NO: 40, SEQ ID NO: 42 and / or SEQ ID NO: 44, but are not limited to this.

[0064] For the purpose of the present disclosure, the microorganism of the present disclosure may include all microorganisms whose formate dehydrogenase activity is weakened compared to its endogenous activity and can produce target purine nucleotides. For example, the microorganism of the present disclosure is characterized in that the activity of formate dehydrogenase is weakened compared to the endogenous activity, thereby increasing the production capacity of purine nucleotides, and may be a transgenic microorganism or a recombinant microorganism, but is not limited thereto. Specifically, the recombinant strain with increased purine nucleotide production capacity may be a microorganism with increased purine nucleotide production capacity compared to a natural wild-type microorganism or an unmodified microorganism with endogenous activity of formate dehydrogenase, but is not limited thereto.

[0065] In one example, the microorganism with purine nucleotide production ability (i.e., the prokaryotic or eukaryotic microbial strain capable of producing purine nucleotides in an organism) may include all microorganisms to which the weakened formate dehydrogenase protein activity of the present application is imparted with purine nucleotide production ability to the microorganism with endogenous purine nucleotide production ability or the parent strain without purine nucleotide production ability. The production ability of purine nucleotides can be imparted or enhanced by the improvement of species.

[0066] "Endogenous activity" refers to the activity of a specific polypeptide possessed by the parent strain before transformation or by an unmodified microorganism when the trait undergoes genetic variation due to natural or human factors. Endogenous activity can also be used interchangeably with "activity before modification".

[0067] As used herein, the term "reducing" polypeptide activity is a comprehensive concept, including reduced activity or no activity compared to its endogenous activity. Reduction can be used interchangeably with terms such as defect, inactivation, deletion, destruction, downregulation, decline, attenuation, inhibition, reduction, etc.

[0068] For example, attenuation may refer to a state in which a protein exhibits activity but is not completely inactivated due to deletion, and may mean that the activity of the protein is attenuated compared to that of an unmodified microorganism, a wild-type strain, or a parent strain, but is not limited thereto.

[0069] For example, attenuation may be, but is not limited to, inactivation. Inactivation may mean that the parent strain or the protein consisting of any one or more amino acid sequences selected from SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and combinations thereof do not express the protein at all compared to the unmodified strain, or even if the protein is expressed, its activity is not expressed or is attenuated.

[0070] Attenuation may also include situations where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide; situations where the overall level of polypeptide activity in the cell is reduced compared to the natural strain due to inhibition of expression of the gene encoding the polypeptide or inhibition of polypeptide translation; situations where the gene is not expressed at all; and situations where no polypeptide activity is observed even if the gene is expressed.

[0071] The reduction of the activity of a polypeptide relative to its endogenous activity means that the activity of the polypeptide is reduced compared to the activity of the initial specific polypeptide possessed by the parent strain or non-modified microorganism before modification. Whether the activity of a polypeptide is reduced can be confirmed from the reduction in the activity level of the corresponding polypeptide, its expression level or the amount of product produced by the corresponding polypeptide.

[0072] In one example, attenuation may mean that the activity of the formate dehydrogenase protein is about less than 100%, about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or 0% relative to the activity of the formate dehydrogenase protein of the parent strain or unmodified microorganism before modification, but is not limited thereto.

[0073] For example, inactivation may mean that the formate dehydrogenase protein is not expressed at all or, if expressed, its activity is not exhibited or is reduced compared to an unmodified microorganism.

[0074] The reduction of polypeptide activity can be carried out by any method known in the art, but the method is not limited thereto, and can be achieved by applying various methods known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0075] Specifically, the attenuation of the polypeptide activity of the present application can be achieved by the following methods:

[0076] (1) Partial or complete deletion of the gene encoding the polypeptide;

[0077] (2) modifying the expression regulatory region (expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced;

[0078] (3) modifying the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is removed or weakened (for example, one or more amino acids are deleted / substituted / added in the amino acid sequence);

[0079] (4) modifying a polynucleotide sequence encoding a polypeptide so that the activity of the polypeptide is removed or weakened (for example, one or more nucleotides in the nucleotide sequence of the polypeptide gene are deleted / replaced / added to encode a polypeptide that has been modified to remove or weaken the activity of the polypeptide);

[0080] (5) modifying the nucleotide sequence of the start codon or 5'-UTR of a gene encoding a polypeptide;

[0081] (6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to a gene transcript encoding a polypeptide;

[0082] (7) adding a sequence complementary to the Shine-Dalgarno (SD) sequence at the front end of the SD sequence of the gene encoding the polypeptide to form a secondary structure, thereby inhibiting ribosome attachment;

[0083] (8) reverse transcription engineering (RTE), which adds a reverse transcription promoter to the 3' end of the open reading frame (ORF) of the polynucleotide sequence encoding the polypeptide; or

[0084] (9) A combination of two or more selected from the above (1) to (8), but not particularly limited thereto.

[0085] For example,

[0086] (1) The method of partially or completely deleting a gene encoding a polypeptide can be achieved by deleting all of the polynucleotide encoding the endogenous target polypeptide in the chromosome, or by replacing the polynucleotide with a polynucleotide having partially deleted nucleotides or a marker gene.

[0087] Partial or complete polynucleotide deletion can be achieved by the following method: by using a vector for inserting into a chromosome in a microorganism for homologous recombination to make the polynucleotide deletion, or by inducing mutations by light (e.g., ultraviolet rays) or chemicals, and selecting a strain with a target gene deletion from the resulting mutant, but not limited thereto. The method of partial or complete gene deletion may include the use of DNA recombination technology. For example, a nucleotide sequence or a vector containing a nucleotide sequence homologous to the target gene may be injected into a microorganism to induce homologous recombination to make partial or complete gene deletion. The injected nucleotide sequence or vector may include a dominant selection marker, but not limited thereto.

[0088] (2) The method of modifying the expression regulatory region can be achieved by inducing modification of the expression regulatory region (expression regulatory sequence) by deletion, insertion, non-conservative substitution or conservative substitution, or a combination thereof; or by replacing the sequence with a sequence with weaker activity. The expression regulatory region may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating transcription and translation, but is not limited thereto.

[0089] The methods of (3) and (4) for modifying an amino acid sequence or a polynucleotide sequence can be achieved as follows: the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide is modified by deletion, insertion, non-conservative or conservative substitution or a combination thereof to reduce the activity of the polypeptide, or the sequence is replaced with an amino acid sequence or polynucleotide sequence modified to have a weaker activity or an amino acid sequence or polynucleotide sequence modified to be inactive, but not limited thereto. For example, by introducing a modification into the polynucleotide sequence to form a stop codon to inhibit or reduce the expression of a gene, but not limited thereto.

[0090] (5) Modification of the nucleotide sequence of the start codon or 5'-UTR of a polypeptide encoding gene can be achieved, for example, by replacing the nucleotide sequence with a nucleotide sequence encoding another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0091] (6) Methods for introducing antisense oligonucleotides (e.g., antisense RNA) that complementarily bind to gene transcripts encoding polypeptides can be found in the literature [Weintraub, H. et alia, Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0092] (7) A sequence complementary to the Shine-Dalgarno (SD) sequence of a gene encoding a polypeptide is added to the front end of the SD sequence to form a secondary structure, thereby inhibiting ribosome attachment, which can be achieved by inhibiting mRNA translation or reducing its rate.

[0093] (8) Reverse transcription engineering (RTE) can be achieved by adding a reverse transcription promoter to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide, by forming antisense nucleotides complementary to the gene transcript encoding the polypeptide to weaken the activity.

[0094] The modification of some or all of the polynucleotides in the microorganism of the present application can be achieved as follows: (a) by homologous recombination through a vector for insertion into the chromosome in the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) by light (e.g., ultraviolet rays and radiation, etc.) and / or chemical treatment induction, but not limited thereto. Methods for modifying some or all of the genes may include methods using DNA recombination technology. For example, by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination, some or all of the genes may be deleted. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

[0095] In one example, the recombinant microorganism having the ability to produce purine nucleotides of the present application may include all microorganisms capable of producing purine nucleotides by reducing the activity of the formate dehydrogenase disclosed in the present invention through vector transformation.

[0096] The vector of the present application may include a DNA construct containing a nucleotide sequence of a polynucleotide encoding a target polypeptide operably connected to a suitable expression control region (expression control sequence) so that the target polypeptide can be expressed in a suitable host cell. The expression control region may include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating transcription and translation termination. Once transferred into a suitable host cell, the vector may be replicated or function independently of the host genome, or may be integrated into the host genome.

[0097] The vector used in this application is not particularly limited, and any vector known in the art can be used. Commonly used vectors include natural or recombinant plasmids, cosmids, viruses and phages. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A can be used; as plasmid vectors, vectors based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH and pET can be used. In particular, vectors such as pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC are also used.

[0098] In one example, the polynucleotide encoding the target polypeptide can be inserted into the chromosome by a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome can be performed by any method known in the art, for example, by homologous recombination, but not limited thereto. The vector may further include a selection marker to confirm the insertion of the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted, and markers providing selectable phenotypes, such as drug resistance, nutritional deficiency, resistance to cytotoxic agents, or expression of surface polypeptides can be used. Only cells expressing the selection marker can survive or show different phenotypes in an environment treated with a selection agent, and therefore the transformed cells can be selected.

[0099] The term "transformation" as used herein refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it does not matter whether the transformed polynucleotide is integrated into the chromosome of the host cell and located inside the host cell or outside the chromosome, and both situations can be included. In addition, the polynucleotide may include DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed therein. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette of a gene structure containing all the elements required for its autonomous expression. The expression cassette can generally include a promoter, a transcription terminator, a ribosome binding site or a translation terminator operably connected to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide can be introduced into the host cell as it is and operably connected to a sequence required for expression in the host cell, but is not limited thereto.

[0100] The term "operably linked" as used herein refers to a structure in which a regulatory sequence is placed in an appropriate position to regulate the expression of a coding sequence. Therefore, the term "operably linked" includes a connection or link between a regulatory region (e.g., a promoter, terminator, signal sequence, or enhancer) having a functional domain of known or desired activity and a target (gene or polypeptide) so that the expression, secretion, or function of the target can be regulated according to the requirements of the known or desired activity. For example, this may mean that a polynucleotide sequence is functionally linked to a promoter sequence that initiates and regulates the transcription of a polynucleotide encoding a target mutant polypeptide of the present application.

[0101] As used herein, the term "expression" includes any step involved in the production of the polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion, but is not limited thereto.

[0102] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule comprising a coding sequence and operably linked regulatory sequences for its expression.

[0103] The term "regulatory sequence" as used herein refers to a polynucleotide sequence necessary for the expression of a coding sequence. Each regulatory sequence may be native (derived from the same source) or foreign (derived from a different gene) to the coding sequence. Examples of regulatory sequences may include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating transcription and translation termination. The smallest unit of a regulatory sequence may include a promoter, and a sequence for terminating transcription and translation.

[0104] The term "recombinant" as used herein with respect to a cell, polynucleotide, polypeptide or vector means that the cell, polynucleotide, polypeptide or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide, or by the alteration of a native polynucleotide or polypeptide, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell may express genes not found in the native (non-recombinant) form of the cell, or may express native genes that are either not expressed at all or expressed abnormally.

[0105] For example, the microorganism used to produce purine nucleotides can be a microorganism that endogenously contains a protein consisting of the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 41 and / or SEQ ID NO: 43, or a protein consisting of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with SEQ ID NO: 39, SEQ ID NO: 41 and / or SEQ ID NO: 43.

[0106] For example, the microorganism for producing purine nucleotides may be an endogenous microorganism comprising a polynucleotide sequence capable of encoding a protein comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 39, SEQ ID NO: 41 and / or SEQ ID NO: 43, a nucleotide sequence of SEQ ID NO: 40, SEQ ID NO: 42 and / or SEQ ID NO: 44, and a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more and less than 100% homology or identity to SEQ ID NO: 40, SEQ ID NO: 42 and / or SEQ ID NO: 44.

[0107] The microorganisms of the present application may include all microorganisms in which the activity of formate dehydrogenase is attenuated compared to its endogenous activity by various methods known in the art.

[0108] In one embodiment, the microorganism with reduced formate dehydrogenase activity compared to its endogenous activity of the present disclosure may be a microorganism in which a polynucleotide encoding formate dehydrogenase is deleted or modified, but is not limited thereto. Specifically, it may be a microorganism without formate dehydrogenase activity due to the deletion of the nucleotide sequence of SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44, but is not limited thereto.

[0109] In one example, the microorganism with increased purine nucleotide production capacity of the present application may be a microorganism with increased purine nucleotide production capacity compared to a non-modified microorganism, but is not limited thereto. In one example, the unmodified microorganism as a target strain for comparing the increased purine nucleotide production capacity may be a CJX1664 strain or a KCCM12151P strain, but is not limited thereto.

[0110] In one example, a microorganism having an increased ability to produce purine nucleotides may have an increase in purine nucleotide production capacity of about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more (the upper limit is not particularly limited, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, or about 40% or less), compared to the parent strain or unmodified microorganism before modification, but is not limited thereto, as long as it has an increased + value compared to the production capacity of the parent strain or non-modified microorganism before modification. In another example, compared to the parent strain or unmodified microorganism before modification, the microorganism with increased purine nucleotide production ability may be about 1.1 times or more, about 1.15 times or more, about 1.2 times or more, about 1.25 times or more, about 1.3 times or more, or about 1.35 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, about 1.5 times or less, about 1.4 times or less), but is not limited thereto.

[0111] In one example, the microorganism with purine nucleotide production capacity can be a prokaryotic cell or a eukaryotic cell, but specifically can be a prokaryotic cell. Prokaryotic cells can include, for example, belonging to Escherichia (Escherichia), Erwinia (Erwinia), Serratia (Serratia), Providencia (Providencia), Corynebacterium (Corynebacterium), Pseudomonas (Pseudomonas), Leptospira (Leptospira), Salmonella (Salmonella), Brevibacteria (Brevibacteria), Hypomononas (Hypomononas), Chromobacterium (Chromobacterium) and Nocardia (Norcardia) microbial strains, or fungi or yeast. Specifically, the microorganism can be a microbial strain belonging to Escherichia (Escherichia), Corynebacterium (Corynebacterium), Leptospira (Leptospira) and Saccharomyces. More specifically, it may be a strain of a microorganism belonging to the genus Corynebacterium.

[0112] According to any of the above embodiments, the microorganism of the present application may be a microorganism belonging to the genus Corynebacterium.

[0113] In one example, the microorganism of the present disclosure may be Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficient, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present disclosure may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium stationis or Corynebacterium glutamicum, but is not limited thereto.

[0114] The microorganism having purine nucleotide-producing ability may be a microorganism having enhanced purine nucleotide-producing ability by further regulating the activity of any one or more proteins selected from the following (a) to (c), but is not limited to:

[0115] (a) the activity of glycine degrading enzyme is enhanced compared with its endogenous activity;

[0116] (b) the serine hydroxymethyltransferase activity is enhanced compared to its endogenous activity; and

[0117] (c) Formate-dependent phosphoribosylglycinamide formyltransferase activity is enhanced compared to its endogenous activity.

[0118] In the microorganism according to any of the above embodiments, the microorganism having the purine nucleotide production ability of the present disclosure may be a microorganism having enhanced purine nucleotide production ability by further regulating the activity of any one or more proteins selected from the following (a) to (d), but is not limited to:

[0119] (a) the activity of glycine degrading enzyme is enhanced compared with its endogenous activity;

[0120] (b) serine hydroxymethyltransferase activity is enhanced compared to its endogenous activity;

[0121] (c) the formate-dependent phosphoribosylglycinamide formyltransferase activity is enhanced compared to its endogenous activity; and

[0122] (d) The serine dehydratase activity is attenuated compared to its endogenous activity.

[0123] The term "enhancement" of polypeptide activity as used herein refers to an increase in the activity of a polypeptide compared to its endogenous activity. Enhancement can be used interchangeably with activation, upregulation, overexpression, increase, and the like.

[0124] Enhancement may include two situations, one of which is the expression of an activity that was not originally possessed, and the other is the enhancement of activity compared to endogenous activity or activity before modification.

[0125] For example, the term "exhibits an activity that was not originally possessed" may refer to "introducing a protein", but is not limited thereto. The introduction of a protein means that a gene that the microorganism did not originally possess is expressed in the microorganism, so that the microorganism exhibits the activity of a specific protein, or exhibits an increase or enhancement in the activity of the corresponding protein compared to the endogenous activity or the activity before modification. For example, the term may mean that a polynucleotide encoding a specific protein is introduced into the chromosome of a microorganism, or a vector containing a polynucleotide encoding a specific protein is introduced into a microorganism, thereby exhibiting the activity of a specific protein.

[0126] "Endogenous activity" refers to the activity of a specific polypeptide possessed by the parent strain before transformation or by an unmodified microorganism when the trait undergoes genetic variation due to natural or human factors. Endogenous activity can also be used interchangeably with "activity before modification".

[0127] An increase in the activity of a polypeptide compared to its endogenous activity means that the activity of the polypeptide is increased compared to the activity and / or concentration (expression level) of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation.

[0128] In one example, enhancement may mean based on the expression of an activity of the corresponding protein that was not originally possessed, or an activity or concentration thereof that is enhanced by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400% or about 500%, up to about 1000% or about 2000% or more than the activity or concentration of the wild-type protein or the initial microbial strain, but is not limited thereto.

[0129] The enhancement of polypeptide activity can be achieved by introducing an exogenous polypeptide or enhancing the activity of an endogenous polypeptide. Whether the activity of a polypeptide is enhanced can be confirmed from the increase in the activity level of the corresponding polypeptide, its expression level or the amount of the product produced by the corresponding polypeptide.

[0130] The enhancement of polypeptide activity can be applied by various methods known in the art, as long as it can enhance the activity of the target polypeptide compared with the microorganism before modification, it is not limited. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art (this is a common method in molecular biology) can be used, but the method is not limited thereto (for example, Sitnicka et alia Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2.1-16, Sambrook et alia Molecular Cloning 2012, etc.).

[0131] Specifically, the enhancement of the polypeptide activity of the present application can be achieved by the following methods:

[0132] (1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide;

[0133] (2) modifying the expression regulatory region of a polypeptide encoding gene on a chromosome (e.g., inducing modification within the expression regulatory region, replacing it with a sequence with stronger activity, or inserting a sequence with stronger activity);

[0134] (3) modifying the nucleotide sequence of the start codon or 5'-UTR of the gene transcript encoding the polypeptide;

[0135] (4) Modifying the amino acid sequence of a polypeptide to enhance the activity of the polypeptide;

[0136] (5) modifying a polynucleotide sequence encoding a polypeptide so that the activity of the polypeptide is enhanced (for example, modifying the polynucleotide sequence of the polypeptide gene so that the polypeptide encoded is modified to have an enhanced polypeptide activity);

[0137] (6) introducing an exogenous polypeptide expressing the activity of the polypeptide or an exogenous polynucleotide encoding the same polypeptide;

[0138] (7) codon optimization of polynucleotides encoding polypeptides;

[0139] (8) analyzing the tertiary structure of the polypeptide to select and modify the exposed sites, or chemically modify them; or

[0140] (9) A combination of two or more selected from the above items (1) to (8), but not particularly limited thereto.

[0141] For example,

[0142] (1) The method of increasing the intracellular copy number of a polynucleotide encoding a polypeptide can be achieved by introducing a vector into a host cell, wherein the vector is operatively linked to the polynucleotide encoding the polypeptide and can replicate and function independently of the host cell. Alternatively, the method can be achieved by introducing one or two copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. Such chromosomal introduction requires a vector that can insert the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

[0143] (2) The method of replacing the expression regulatory region (or expression regulatory sequence) of the gene encoding the polypeptide on the chromosome with a sequence having strong activity is achieved, for example, by inducing modification of the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacing the sequence with a sequence having stronger activity to further enhance the activity of the expression regulatory region. The expression regulatory region may include but is not particularly limited to a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination, etc. In one example, the method may include replacing the original promoter with a strong promoter, but is not limited thereto.

[0144] Examples of known strong promoters may include cj1 to cj7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US10584338 B2), O2 promoter (US10273491 B2), tkt promoter, yccA promoter, etc., but the strong promoter is not limited thereto.

[0145] (3) A method of modifying the nucleotide sequence of the start codon or 5′-UTR of the gene encoding the polypeptide, for example, by replacing the nucleotide sequence with a nucleotide sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but not limited thereto.

[0146] (4) and (5) Modification of the amino acid sequence or polynucleotide sequence The amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide can be modified by deletion, insertion, non-conservative or conservative substitution or a combination thereof to enhance the activity of the polypeptide; or the sequence can be replaced with an amino acid sequence or polynucleotide sequence modified to have a stronger activity or an amino acid sequence or polynucleotide sequence modified to enhance the activity, but is not limited thereto. The replacement can be specifically carried out by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used in this article may further include a selection marker for confirming the insertion into the chromosome. The selection marker is as described above.

[0147] (6) The method of introducing an exogenous polynucleotide that exhibits the activity of the polypeptide can be achieved by introducing an exogenous polynucleotide encoding a polypeptide that exhibits the same / similar activity as the polypeptide into a host cell. The exogenous polynucleotide can be used without restriction, regardless of its source or sequence, as long as it exhibits the same / similar activity as the polypeptide. The introduction can be performed by a person of ordinary skill in the art by selecting an appropriate transformation method known in the art, and the expression of the introduced polynucleotide in the host cell can produce the polypeptide, thereby increasing its activity.

[0148] (7) The codon optimization method of the polynucleotide encoding the polypeptide can be achieved by codon optimization of the endogenous polynucleotide to increase transcription or translation in the host cell, or by optimizing its codons so that optimized transcription and translation of the exogenous polynucleotide can be achieved in the host cell.

[0149] (8) A method for analyzing the tertiary structure of a polypeptide to select and modify an exposure site, or to chemically modify it, can be achieved, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, and then determining a candidate template protein based on sequence similarity, and confirming the structure based on the sequence information, thereby selecting and modifying or modifying the exposure site to be modified or chemically modified.

[0150] Such enhancement of polypeptide activity may mean that the activity and concentration of the corresponding polypeptide are increased relative to the activity or concentration of the polypeptide expressed in the wild type or microbial strain before modification, or the amount of the product produced by the corresponding polypeptide is increased, but is not limited thereto.

[0151] In one example, the microorganism having the purine nucleotide-producing ability of the present disclosure may be a microorganism having enhanced purine nucleotide-producing ability by further enhancing the activity of glycine degrading enzyme relative to its endogenous activity, but is not limited thereto.

[0152] The term "glycine degrading enzyme" as used herein is a protein that directly or indirectly participates in the glycine degradation pathway, and can be used to represent each protein constituting the glycine degradation system, or a protein complex or the glycine degradation system itself. Specifically, the glycine degrading enzyme can be selected from any one or more proteins of the T protein (GcvT), P protein (GcvP), L protein (GcvL), H protein (GcvH) and the coenzyme LipB or LipA of the glycine degradation system, but is not limited thereto (John E. Cronan, Microbiology and Molecular Biology Reviews, 13 April 2016). The glycine degrading enzyme of the present application can be used interchangeably with GcvPTH or the glycine cleavage system.

[0153] The term "glycine cleavage system (GCV)" as used herein is composed of two or more subunits, namely, subunits GcvP, GcvT and GcvH, which catalyze the oxidative decarboxylation and deamination of glycine to carbon dioxide, ammonium ions and N 5-10 - The form of a multi-enzyme complex of methylenetetrahydrofolate. The glycine cleavage system of the present application can be used interchangeably with the glycine degradation system.

[0154] Specifically, the glycine degrading enzyme of the present application may be a protein having the activity of GcvP, GcvT and GcvH proteins encoded by gcvP, gcvT and gcvH genes, respectively, and its type is not particularly limited as long as it has the activity corresponding to the glycine degrading enzyme. The GcvP, GcvT and GcvH proteins encoded by the gcvP, gcvT and gcvH genes, respectively, are known in the art, and the amino acid and polynucleotide sequences of each GcvP, GcvT and GcvH protein can be obtained from known databases, such as GenBank of NCBI, etc., but are not limited thereto.

[0155] In one example, the GcvP, GcvT and GcvH proteins of the present disclosure may respectively comprise the amino acid sequences of SEQ ID NO: 47, SEQ ID NO: 49 and SEQ ID NO: 51, or amino acid sequences having at least 60% homology or identity thereto, but are not limited thereto as long as they have the activity of a glycine degrading enzyme. Specifically, the polypeptides having the activity of the GcvP, GcvT and GcvH proteins may respectively have or comprise the amino acid sequences of SEQ ID NO: 47, SEQ ID NO: 49 and SEQ ID NO: 51, or amino acid sequences having at least 60%, 70%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98% or 99% homology or identity thereto, or consist of or consist essentially of. In one example, GcvP, GcvT and GcvH proteins may refer to proteins endogenously present in Corynebacterium microorganisms or Corynebacterium stationis, but are not limited thereto. Specifically, GcvP, GcvT and GcvH proteins may be GcvP proteins consisting of the amino acid sequence of SEQ ID NO: 47, GcvT proteins consisting of the amino acid sequence of SEQ ID NO: 49, and GcvH proteins consisting of the amino acid sequence of SEQ ID NO: 51, respectively, endogenously present in Corynebacterium microorganisms or Corynebacterium stationis, but are not limited thereto.

[0156] Furthermore, the nucleotide sequences encoding the GcvP, GcvT and GcvH proteins may be nucleotide sequences encoding proteins exhibiting glycine degrading enzyme activity.

[0157] For example, the GcvP, GcvT and GcvH proteins having the amino acid sequences of SEQ ID NO: SEQ ID NO: 47, SEQ ID NO: 49 and SEQ ID NO: 51, respectively, may be encoded by a polynucleotide having or comprising the nucleotide sequences of SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 52, or nucleotide sequences having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more and less than 100% homology or identity with SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 52, or consisting of or consisting essentially of, but not limited to, the nucleotide sequences of SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 52 may be obtained from known databases, such as GenBank of NCBI, etc., but not limited to, these.

[0158] In one example, the microorganism having the purine nucleotide-producing ability of the present disclosure may be a microorganism having enhanced purine nucleotide-producing ability by further enhancing the activity of serine hydroxymethyltransferase relative to its endogenous activity, but is not limited thereto.

[0159] The term "serine hydroxymethyltransferase" used herein is an enzyme that catalyzes the conversion of serine into glycine. The serine hydroxymethyltransferase of the present application can be used interchangeably with GlyA. Specifically, the serine hydroxymethyltransferase of the present application can be a protein with serine hydroxymethyltransferase activity encoded by the glyA gene, but as long as the protein has an activity corresponding to the serine hydroxymethyltransferase, its type is not particularly limited. The serine hydroxymethyltransferase encoded by the glyA gene is well known in the art, and the amino acid and polynucleotide sequences of the serine hydroxymethyltransferase can be obtained from the known database NCBI's GenBank, etc., but are not limited thereto.

[0160] In one example, the serine hydroxymethyltransferase may comprise the amino acid sequence of SEQ ID NO: 53 or an amino acid sequence having 60% or more homology thereto, but is not limited thereto as long as it has the activity of the serine hydroxymethyltransferase. Specifically, the polypeptide having the serine hydroxymethyltransferase activity may have or comprise the amino acid sequence of SEQ ID NO: 53 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98% or 99% homology or identity thereto, or consist of or consist essentially of. In one example, the serine hydroxymethyltransferase may refer to a protein endogenously present in a microorganism of the genus Corynebacterium or Corynebacterium stationis, but is not limited thereto. Specifically, it may be a serine hydroxymethyltransferase consisting of the amino acid sequence of SEQ ID NO: 53 which is endogenously present in a Corynebacterium microorganism or Corynebacterium stationis, but is not limited thereto.

[0161] Furthermore, the nucleotide sequence encoding serine hydroxymethyltransferase may be a nucleotide sequence encoding a protein exhibiting serine hydroxymethyltransferase activity.

[0162] For example, the serine hydroxymethyltransferase having the amino acid sequence of SEQ ID NO: 53 may be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 54, or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity with SEQ ID NO: 54, or consisting of or consisting essentially of, but not limited to, the nucleotide sequence of SEQ ID NO: 54 may be obtained from known databases, such as GenBank of NCBI, etc., but not limited to, the nucleotide sequence of SEQ ID NO: 54.

[0163] In one example, the microorganism having the purine nucleotide production ability of the present disclosure may be a microorganism having enhanced purine nucleotide production ability by further enhancing the activity of formate-dependent phosphoribosylamide formyltransferase relative to its endogenous activity, but is not limited thereto.

[0164] The term "formate-dependent phosphoribosylglycinamide formyltransferase" as used herein has the activity of catalyzing the following chemical reaction: The formate-dependent phosphoribosylglycine formyltransferase of the present application can be used interchangeably with PurT. Specifically, the formate-dependent phosphoribosylglycine formyltransferase of the present application can be a protein having formate-dependent phosphoribosylglycine formyltransferase activity encoded by the purT gene, but as long as the protein has an activity corresponding to formate-dependent phosphoribosylglycine formyltransferase, its type is not particularly limited. The formate-dependent phosphoribosylglycine formyltransferase encoded by the purT gene is known in the art, and the amino acid and polynucleotide sequences of the formate-dependent phosphoribosylglycine formyltransferase can be obtained from the known database NCBI's GenBank, etc., but are not limited thereto.

[0165] In one example, the formate-dependent phosphoribosylglycinamide formyltransferase may comprise the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having 60% or more homology thereto, but is not limited thereto as long as it has the activity of formate-dependent phosphoribosylglycinamide formyltransferase. Specifically, the polypeptide having formate-dependent phosphoribosylglycinamide formyltransferase activity may have or comprise the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity thereto, or may consist of or consist essentially of the same. In one example, the formate-dependent phosphoribosylglycinamide formyltransferase may refer to a protein endogenously present in a microorganism of the genus Corynebacterium or Corynebacterium stationis, but is not limited thereto. Specifically, it may be a formate-dependent phosphoribosylglycinamide formyltransferase endogenously present in a Corynebacterium microorganism or Corynebacterium stationis consisting of the amino acid sequence of SEQ ID NO: 55, but is not limited thereto.

[0166] Furthermore, the nucleotide sequence encoding formate-dependent phosphoribosylglycinamide formyltransferase may be a nucleotide sequence encoding a protein exhibiting formate-dependent phosphoribosylglycinamide formyltransferase activity.

[0167] For example, the formate-dependent phosphoribosylglycinamide formyltransferase having the amino acid sequence of SEQ ID NO: 55 may be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 56 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity with SEQ ID NO: 56, or consisting of or consisting essentially of, but not limited to, the nucleotide sequence of SEQ ID NO: 56 can be obtained from known databases, such as GenBank of NCBI, etc., but not limited to, the nucleotide sequence of SEQ ID NO: 56.

[0168] In one example, the microorganism having the purine nucleotide-producing ability of the present disclosure may be a microorganism having enhanced purine nucleotide-producing ability by further weakening the activity of serine dehydratase relative to its endogenous activity, but is not limited thereto.

[0169] The term "serine dehydratase (L-serine dehydratase)" as used herein is a The serine dehydratase disclosed herein can be used interchangeably with SdaA or L-serine ammonia lyase. Specifically, the serine dehydratase of the present application can be a protein having serine dehydratase activity encoded by the sdaA gene, and its type is not particularly limited as long as the protein has an activity corresponding to the serine dehydratase. The serine dehydratase encoded by the sdaA gene is known in the art, and the amino acid and polynucleotide sequences of the serine dehydratase can be obtained from the known database NCBI's GenBank, etc., but are not limited thereto.

[0170] In one example, the serine dehydratase may comprise the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having more than 60% homology thereto, but is not limited thereto as long as it has the activity of the serine dehydratase. Specifically, the polypeptide having the serine dehydratase activity may have or comprise the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98% or 99% homology or identity thereto, or be composed of or substantially composed of the same. In one example, the serine dehydratase may refer to a protein endogenously present in a Corynebacterium microorganism or Corynebacterium stationis, but is not limited thereto. Specifically, it may be a serine dehydratase consisting of the amino acid sequence of SEQ ID NO: 45 which is endogenously present in a Corynebacterium microorganism or Corynebacterium stationis, but is not limited thereto.

[0171] Furthermore, the nucleotide sequence encoding the serine dehydratase may be a nucleotide sequence encoding a protein exhibiting serine dehydratase activity.

[0172] For example, the serine dehydratase having the amino acid sequence of SEQ ID NO: 45 may be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 46 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity with SEQ ID NO: 46, or consisting of or consisting essentially of, but not limited to, the nucleotide sequence of SEQ ID NO: 46 may be obtained from known databases, such as GenBank of NCBI, etc., but not limited to, the nucleotide sequence of SEQ ID NO: 46.

[0173] In the microorganism according to any of the above embodiments, the microorganism having the purine nucleotide production ability disclosed in the present invention may be a microorganism having enhanced purine nucleotide production ability by weakening the activity of formate dehydrogenase relative to its endogenous activity and enhancing the activity of formate ribosylglycinamide formyltransferase relative to its endogenous activity, but is not limited thereto.

[0174] In the microorganisms according to any of the above embodiments, the microorganism having the purine nucleotide production ability disclosed in the present invention may be a microorganism having enhanced purine nucleotide production ability by weakening the activity of formate dehydrogenase relative to its endogenous activity, enhancing the activity of formate ribose-dependent phosphoribosylamide formyltransferase relative to its endogenous activity, and enhancing the activity of glycine degrading enzyme compared to its endogenous activity, but is not limited thereto.

[0175] According to the microorganism of any of the above-mentioned embodiments, the microorganism having the ability to produce purine nucleotides of the present application may be a microorganism having enhanced purine nucleotide production ability by weakening the serine dehydratase activity compared with its endogenous activity, enhancing the serine hydroxymethyltransferase activity compared with its endogenous activity, enhancing the glycine degrading enzyme activity compared with its endogenous activity, weakening the formate dehydrogenase activity compared with its endogenous activity, and enhancing the formate-dependent phosphoribosylglycinamide formyltransferase activity compared with its endogenous activity, but is not limited thereto.

[0176] Another aspect of the present application provides a method for producing purine nucleotides, comprising: culturing the microorganism of the present application in a culture medium, wherein the activity of formate dehydrogenase of the microorganism is weakened relative to its endogenous activity.

[0177] As used herein, the term "culture" refers to growing the strain of the present application under suitably controlled environmental conditions. The culture process of the present application can be carried out under suitable culture media and culture conditions known in the art. Those skilled in the art can easily adjust and use such culture processes according to the selected strain. Specifically, the culture can be batch culture, continuous culture and / or fed-batch culture, but is not limited thereto.

[0178] Term used herein " culture medium " refers to contain the mixture of the material of the required nutrient substance of cultivating the microorganism of the present application as main component, and it provides nutrient substance and growth factor, and the necessary water of survival and growth.Specifically, the culture medium and other culture conditions for cultivating the bacterial strain of the present application can be any culture medium for the conventional cultivation of microorganisms, without any particular restrictions.But the microorganism of the present application, in the conventional culture medium containing suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, under aerobic conditions, regulates temperature, pH etc. simultaneously and cultivates.For example, the culture medium of Corynebacterium (Corynebacterium) microorganism can be found in the literature [" Manual of Methods for General Bacteriology " by the American Society for Bacteriology (Washington D.C., USA, 1981)].

[0179] In the present application, the carbon source may include carbohydrates such as glucose, sugar, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, the carbon source may include natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, cane molasses, and corn steep liquor, etc. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used, and in addition, various other carbon sources can be used in appropriate amounts without restriction. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0180] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0181] The phosphorus source may include monopotassium phosphate, dipotassium phosphate or corresponding sodium salts, etc. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins and / or appropriate precursors, etc. may also be included. These constituents or precursors may be added to the culture medium in batches or continuously, but these phosphorus sources are not limited thereto.

[0182] In addition, the pH value of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. in an appropriate manner during the microbial culture process of the present application. In addition, defoamers (such as fatty acid polyethylene glycol esters) can be used to prevent bubble formation during the culture process. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain aerobic conditions of the culture medium; or nitrogen, hydrogen or carbon dioxide can be injected to maintain anaerobic conditions or microaerobic conditions without injecting gas, but the gas is not limited thereto.

[0183] The temperature during the culture process of the present application may be in the range of 27° C. to 37° C., specifically 30° C. to 33° C., and the culture may last for 20 to 120 hours, but is not limited thereto.

[0184] The term "culture product" as used herein refers to a culture solution, a concentrated culture solution, a dried product of the culture solution, a culture filtrate, a concentrated culture filtrate, or a dried product of the concentrated filtrate obtained by culturing a specific microorganism in a culture medium, and means that the culture solution may include the specific microorganism, while the culture filtrate substantially does not include the specific microorganism (particularly, it substantially means excluding the specific microorganism separated by filtration, etc., but does not mean that the microorganism is completely excluded from the filtrate). The dosage form of the culture product is not limited and may be, for example, a liquid, an emulsion, or a solid. Specifically, for the purpose of the present application, the culture product may include purine nucleotides.

[0185] The term "fermentation" as used herein refers to the process in which microorganisms use their own enzymes to decompose organic matter without causing decay. The processes of fermentation and decay reactions are similar, but when the decomposition produces useful substances, it is called fermentation, and when it produces smelly or harmful substances, it is called decay.

[0186] In the present application, the method for obtaining the fermentation product from the strain is not particularly limited, and can be obtained according to methods commonly used in this field or similar fields.

[0187] As used herein, the term "fermentation product" may include not only the fermentation substance itself, but also all kinds of substances, including the fermentation product produced from the strain, such as the culture medium of the strain in which the strain coexists with the culture, the fermentation product produced by the culture medium, the fermentation product obtained by sterilizing the strain from the culture medium, the fermentation product obtained by sterilizing the strain from the culture medium and filtering the strain, the extract obtained by extracting the fermentation product or the culture medium containing the fermentation product, the diluted solution obtained by diluting the fermentation product or its extract, the concentrated solution, the dried product obtained by drying the fermentation product or its extract, and the lysate obtained by collecting and lysing the strain cells, etc.

[0188] In the method of the present application, the culture of the microorganism can be carried out by any culture conditions and culture methods known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the selected strain.

[0189] The purine nucleotides produced by the culture of the present application may be released into the medium or retained in the cells.

[0190] In one embodiment, the method for producing purine nucleotides of the present application may also, for example, include a step of preparing the microorganism of the present application, a step of preparing a culture medium for culturing the strain, or a combination thereof (regardless of the order, in any order) before the culturing step.

[0191] The method for producing purine nucleotides of the present application may further include recovering the target substance, specifically, purine nucleotide, from the cultured microorganism, the culture product of the microorganism, the fermentation product of the microorganism or the culture medium. The culturing step may further include a recovery step.

[0192] In the recovery step, the culture method of the microorganism of the present application (for example, according to batch culture, continuous culture or fed-batch culture method) can be used to collect the target purine nucleotide using a suitable method known in the art. For example, centrifugation, filtration, treatment with a protein crystallization precipitant (salting out), extraction, ultrasonic crushing, ultrafiltration, dialysis, various chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography), HPLC and other methods or a combination of the above methods can be used, and the target substance, particularly purine nucleotides, can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0193] In addition, the production method of purine nucleotides of the present application may further include a purification step, which may be performed using an appropriate method known in the art. In one example, when the production method of purine nucleotides of the present application includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or intermittently, or simultaneously, or may be integrated into one step, but the method is not limited thereto.

[0194] In the method of the present disclosure, the attenuation of formate dehydrogenase activity and the purine nucleotides are as described in other aspects above.

[0195] Another aspect of the present application provides a composition for producing purine nucleotides, comprising: the microorganism disclosed herein, wherein the activity of formate dehydrogenase is weakened relative to its endogenous activity, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof.

[0196] The composition of the present application may also include any suitable excipient commonly used in compositions for producing purine nucleotides, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, etc., but are not limited thereto.

[0197] In one embodiment, each component present in the composition of the present application may contain a microbiologically effective amount, or may be present in the composition in an appropriate amount for production.

[0198] In the composition of the present disclosure, the attenuation of formate dehydrogenase activity and purine nucleotides are as described in other aspects above.

[0199] Another aspect of the present application provides use of the microorganism of the present application for producing purine nucleotides, wherein the formate dehydrogenase activity of the microorganism is weakened compared to its endogenous activity.

[0200] In the use of the present disclosure, the attenuation of formate dehydrogenase activity and purine nucleotides are as described in other aspects above.

[0201] [Implementation Methods of the Invention]

[0202] Hereinafter, the present application will be described in detail by way of examples. However, these examples are preferred examples given for illustrative purposes only, and therefore, the scope of the present application is not limited to or restricted by these examples. Meanwhile, the technical features not described herein can be fully understood and easily performed by those skilled in the art in the technical field of the present application or similar technical fields.

[0203] [Example 1. Preparation of formate dehydrogenase-deficient strains, preparation of formate-dependent phosphoribosylglycinamide formyltransferase-enhanced strains and evaluation of IMP production capacity]

[0204] [Example 1-1. Preparation of formate dehydrogenase-deficient recombinant vector and IMP production strain]

[0205] The formate dehydrogenase derived from Corynebacterium stationis may be composed of formate dehydrogenase subunit 1 (Fdh subunit 1), formate dehydrogenase subunit 2 (Fdh subunit 2) and formate dehydrogenase subunit 3 (Fdh subunit 3).

[0206] An experiment was conducted to prepare a strain in which endogenous formate dehydrogenase was deleted in Corynebacterium stationis KCCM12151P (IMP-producing strain) to confirm whether increasing formate by deleting formate dehydrogenase subunit 1, formate dehydrogenase subunit 2 and formate dehydrogenase subunit 3 (hereinafter referred to as formate dehydrogenase) would lead to an increase in IMP production.

[0207] Specifically, an experiment was conducted to prepare a strain in which the fdh1, fdh2, and fdh3 genes (hereinafter referred to as fdh genes) encoding formate dehydrogenase subunit 1, formate dehydrogenase subunit 2, and formate dehydrogenase subunit 3, respectively, were deleted.

[0208] Use G-spin total DNA extraction mini kit (Cat.No.17045, Intron) to separate the chromosomal gene of ATCC6872 strain (wild-type Corynebacterium stationis) according to the scheme provided in the kit, use the primer pair of SEQ ID NO:1 and 2 and the primer pair of SEQ ID NO:3 and 4 to carry out PCR, obtain a pair of gene fragments (fdh-A, fdh-B) respectively.PCR is carried out under the following conditions: denaturation at 94 ℃ for 5min, then 20 cycles (denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, polymerization at 72 ℃ for 1 minute), then polymerization at 72 ℃ for 7min.As a result, polynucleotides of 1091bp and 1105bp were obtained respectively.

[0209] The two fragments were used as templates for secondary PCR to obtain a 2.2 kbp polynucleotide template. The obtained gene fragment was cut with the restriction endonuclease XbaI (New England Biolabs, Beverly, MA). The gene fragment cut with the XbaI restriction endonuclease was connected to the linear pDCM2 (Korean Patent Publication No. 10-2020-0136813) using T4 ligase (New England Biolabs, Beverly, MA). The resulting vector was named pDCM2-del_fdh.

[0210] The primer sequences used for vector preparation are as follows.

[0211]

Table 1

[0212] SEQ ID NO: name sequence 1 fdh-A_F 5'-AATTCGAGCT CGGTACCCGT TGCCGTATCA GACATGCT-3' 2 fdh-AR 5'-TCATAGGTGC CGAACTTGAT TTATCTCGAC CAAAC-3' 3 fdh-BF 5'-GTCGAGATAAATCAAGTTCGGCACCTATGAGAATA-3' 4 fdh-BR 5'-CGACTCTAGA GGATCCCCCT CGGGTTCCAT GTTGTCTT-3'

[0213] The pDCM2-del_fdh vector was transformed into the bacterial strain Corynebacterium stationis KCCM12151P (IMP production strain) with IMP production ability by electroporation, and then the bacterial strain with the mutant gene and the carrier inserted on the chromosome was selected as a preliminary candidate strain in the selection medium containing 25mg / L kanamycin. After that, the endogenous formate dehydrogenase on the chromosome was deleted by the secondary crossover process utilizing the homology between the gene present on the chromosome and the gene inserted by the vector, and the final bacterial strain from which the carrier containing the kanamycin resistance gene was removed was obtained. The deletion strain was first confirmed by PCR utilizing SEQ ID NO: 5 and 6 primers, and finally confirmed by gene sequencing analysis. The formate dehydrogenase-deficient strain obtained by the above method was named CJI-3162 (KCCM12151P_fdh_deficient) and was deposited in the Korean Collection of Microorganisms (KCCM), a depository under the Budapest Treaty, on November 24, 2022, with the deposit number KCCM13275P.

[0214]

Table 2

[0215] SEQ ID NO: name sequence 5 fdh-deletion-confirmation_F 5'-CTTGTGCGCC GAAGCCCGTT-3' 6 fdh-deletion-confirmation_R 5'-GCTTTCATGA GCTTGCCGGG-3'

[0216] [Example 1-2. Preparation of formate dehydrogenase-deficient and formate-dependent phosphoribosylglycinamide formyltransferase-enhanced integrated strains and IMP-producing strains]

[0217] Strains incorporating a deletion of formate dehydrogenase and an enhancement of formate-dependent phosphoribosylglycinamide formyltransferase were generated and evaluated.

[0218] In order to prepare a strain in which the activity of the endogenous purT gene was additionally enhanced in the CJI-3162 strain produced in Example 1-1, an experiment was conducted to enhance purT in the form of a Pcj7 promoter (Korean Application No. 10-2004-0107215).

[0219] Use G-spin total DNA extraction mini kit (Cat.No.17045, Intron) to separate the chromosomal gene of ATCC6872 strain (wild-type Corynebacterium stationis) according to the scheme provided in the kit, use the primer pair of SEQ ID NO:7 and 8, the primer pair of SEQ ID NO:9 and 10 and the primer pair of SEQ ID NO:11 and 12 to carry out PCR, obtain a pair of gene fragments (Pcj7 / purT-A, Pcj7 / purT-B, Pcj7 / purT-C). PCR is carried out under the following conditions: denaturation at 94°C for 5min, then 20 cycles (denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute), and then polymerization at 72°C for 7min. As a result, polynucleotides of 851bp, 318bp and 882bp were obtained respectively.

[0220] The three fragments were used as templates for secondary PCR to obtain a 2 kbp polynucleotide template. The obtained gene fragment was cut with the restriction endonuclease XbaI (New England Biolabs, Beverly, MA). The gene fragment cut with the XbaI restriction endonuclease was ligated to the linearized pDCM2 (Korean Patent Publication No. 10-2020-0136813) using T4 ligase (New England Biolabs, Beverly, MA). The resulting vector was named pDCM2-Pcj7 / purT.

[0221] The primer sequences used for vector preparation are as follows.

[0222]

Table 3

[0223]

[0224] The pDCM2-Pcj7 / purT vector was transformed into CJI-3162 (KCCM12151P_fdh_ deleted) prepared in Example 1-1 by electroporation, and then the strain with the mutant gene and the vector inserted on the chromosome was selected as a preliminary candidate in a selection medium containing 25 mg / L kanamycin. Thereafter, the endogenous purT promoter was enhanced with Pcj7 through a secondary crossover process using the homology between the gene present on the chromosome and the gene inserted by the vector, and the final strain containing the vector containing the kanamycin resistance gene was removed therefrom. The purT enhanced strain was first confirmed by PCR using SEQ ID NO: 13 and 14 primers, and finally confirmed by gene sequencing analysis. The Pcj7 / purT strain obtained by the above method was named CJI-3163 (CJI3162_Pcj7 / purT).

[0225]

Table 4

[0226] SEQ ID NO: name sequence 13 Pcj7 / purT-Confirm_F 5'-CGAATACCTC CAGACCACCG-3' 14 Pcj7 / purT-Confirm_R 5'-TGGGCACAAC GGTGACAGCG-3'

[0227] [Example 1-3. Evaluation of IMP production capacity of two strains: a formate dehydrogenase-deficient strain and a formate-dependent phosphoribosylglycinamide formyltransferase-enhanced strain]

[0228] The IMP production capacity of the above strains was evaluated by flask evaluation. Corynebacterium stationis KCCM12151P, CJI-3162 and CJI-3163 were inoculated into a 14mL test tube containing less than 2.5mL of seed culture medium and incubated for 24 hours at 30°C and 170rpm. Then, 2mL of seed culture solution was inoculated into a 250mL corner baffle flask containing less than 29mL of production culture medium (24mL main culture medium + 5mL separate sterile culture medium), and incubated for 72 hours with shaking at 30°C and 170rpm. After the cultivation is completed, the IMP production capacity is measured by HPLC.

[0229] The compositions of the seed medium and fermentation medium are as follows.

[0230]

IMP seed culture medium

[0231] Glucose 1%, peptone 1%, meat extract 1%, yeast extract 1%, sodium chloride 0.25%, adenine 100mg / L, guanine 100mg / L, pH 7.2

[0232]

IMP flask fermentation medium

[0233] Sodium glutamate 0.1%, ammonium chloride 1%, magnesium sulfate 1.2%, calcium chloride 0.01%, iron sulfate 20mg / L, manganese sulfate 20mg / L, zinc sulfate 20mg / L, copper sulfate 5mg / L, L-cysteine ​​23mg / L, β-alanine 24mg / L, niacin 8mg / L, biotin 45μg / L, thiamine hydrochloride 5mg / L, adenine 30mg / L, phosphoric acid (85%) 1.9%, glucose 4.2%, fructose 2.4%

[0234] The culture results of Corynebacterium stationis according to the deletion of formate dehydrogenase and the enhancement of formate-dependent phosphoribosylamide formyltransferase are shown in Table 5 below.

[0235] [Table 5: Confirmation of IMP production by formate dehydrogenase deficiency and formate-dependent phosphoribosylglycinamide formyltransferase enhancement]

[0236] Strain number Import Type OD IMP(g / L) Concentration increase rate (%) KCCM12151P Control group 40.3 5.4 - CJI-3162 △fdh 41.3 5.8 7.4 CJI-3163 CJI03162-Pcj7 / purT 40.8 6.1 12.9

[0237] The IMP production capacity of the fdh-deficient strain among the IMP-producing strains was increased by 7.4% compared with the control group, and it was confirmed that when the purT gene in the fdh-deficient strain was enhanced, the IMP production capacity was further enhanced.

[0238] [Example 2. Preparation and evaluation of gcvPTH-enhanced, sdaA-deficient and glyA-enhanced strains based on formate dehydrogenase-deficient and formate-dependent phosphoribosylamide formyltransferase-enhanced strains]

[0239] [Example 2-1. Preparation of gcvPTH gene-enhanced recombinant vector]

[0240] Added a copy of gcvPTH.

[0241] A vector to which a copy of the endogenous gcvPTH gene of the Corynebacterium stationis strain was added in the form of a natural promoter was prepared as follows using plasmid pDCM2 (Korean Patent Publication No. 10-2020-0136813) for gene insertion and replacement into the chromosome of Corynebacterium.

[0242] The chromosomal gene of ATCC6872 strain (wild-type Corynebacterium stationis) was isolated using a G centrifugal total DNA extraction mini kit (Cat. No. 17045, Intron) according to the protocol provided in the kit, and PCR was performed using primer pairs of SEQ ID NOs: 15 and 16, primer pairs of SEQ ID NOs: 17 and 18, primer pairs of SEQ ID NOs: 19 and 20, and primer pairs of SEQ ID NOs: 21 and 22 to obtain gene fragments (Pn / gcvPTH-A, Pn / gcvPTH-B, Pn / gcvPTH-C, Pn / gcvPTH-D), respectively. PCR was performed under the following conditions: denaturation at 94°C for 5 min, followed by 20 cycles (denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute), and then polymerization at 72°C for 7 min.

[0243] The obtained gene fragment was cut with restriction endonuclease XbaI (New England Biolabs, Beverly, MA). The gene fragment cut with XbaI restriction endonuclease was ligated to linear pDCM2 (Korean Patent Publication No. 10-2020-0136813) using T4 ligase (New England Biolabs, Beverly, MA). The resulting vector was named pDCM2-Pn / gcvPTH.

[0244] The primer sequences used for vector preparation are as follows.

[0245]

Table 6

[0246]

[0247]

[0248] [Example 2-2. Preparation of IMP-producing strain with additional enhancement of gcvPTH gene]

[0249] An experiment was conducted to further enhance gcvPTH in CJI-3163 (CJI3162_Pcj7 / purT) prepared in the above Example 1-2. The pDCM2-Pn / gcvPTH vector prepared in Example 2-1 was transformed into CJI-3163 (CJI3162_Pcj7 / purT) prepared in Example 1-2 by electroporation, and then the strains with mutant genes and vectors inserted on the chromosome were screened as preliminary candidate strains in a selection medium containing 25 mg / L kanamycin. Thereafter, in the strains where homologous recombination occurred, it was first confirmed by PCR using SEQ ID NO: 23 and 24 primer pairs, and finally confirmed by gene sequencing analysis. The selected strain was named CJI-3188 (CJI-3163_Pn / gcvPTH).

[0250]

Table 7

[0251] SEQ ID NO: name sequence 23 Primers 5'-CCAGTACATT GTCATGGGAT-3' 24 Primers 5'-GATTGGCATA TCGCACCGAG-3'

[0252] [Example 2-3. Preparation of sdaA-deleted and glyA-added enhanced recombinant vector and preparation of IMP production strain]

[0253] The vector was prepared in such a manner that the endogenous gene sdaA of Corynebacterium stationis was deleted and the copy number of the glyA gene was added at the corresponding position.

[0254] The chromosomal genes of ATCC6872 strain (wild-type Corynebacterium stationis) were isolated using the G-spin total DNA extraction mini kit (Cat. No. 17045, Intron) according to the protocol provided in the kit, and PCR was performed using primer pairs of SEQ ID NOs: 25 and 26, primer pairs of SEQ ID NOs: 27 and 28, primer pairs of SEQ ID NOs: 29 and 30, and primer pairs of SEQ ID NOs: 31 and 32 to obtain gene fragments (del_sda::Pcj7 / glyA-A, del_sda::Pcj7 / glyA-B, del_sda::Pcj7 / glyA-C, del_sda::Pcj7 / glyA-D). PCR was performed under the following conditions: denaturation at 94°C for 5 min, followed by 20 cycles (denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, polymerization at 72°C for 1 min), and then polymerization at 72°C for 7 min.

[0255] The gene fragment obtained by secondary PCR using the four fragments obtained as above as templates was cut with restriction endonuclease XbaI (New England Biolabs, Beverly, MA). The gene fragment cut with XbaI restriction endonuclease was connected to linear pDCM2 (Korean Patent Publication No. 10-2020-0136813) using T4 ligase (New England Biolabs, Beverly, MA). The resulting plasmid was named pDCM2-del_sdaA-Pn / glyA.

[0256] The sequences of the primers used for vector preparation are as follows.

[0257]

Table 8

[0258]

[0259]

[0260] The pDCM2-del_sdaA-Pcj7 / glyA vector was transformed into the CJI-3188 strain (CJI-3163_Pn / gcvPTH) prepared in Example 2-2 by electroporation, and then the strain with the mutant gene and the vector inserted on the chromosome was selected as the preliminary candidate strain in the selection medium containing 25 mg / L kanamycin. Thereafter, among the strains in which homologous recombination occurred, the primer pairs of SEQ ID NO: 34 and 35 were used to select the strain in which sdaA was deleted and the glyA gene connected to the Pcj7 promoter was inserted at the corresponding position. The strain obtained by the above method was named CJI-3189 (CJI-3188_del_sdaA-Pn / glyA).

[0261] The sequences of the primers used for strain preparation are as follows.

[0262]

Table 9

[0263] SEQ ID NO: name sequence 33 Primers 5'-TAATCCCCCA GCTCACCGGC-3' 34 Primers 5'-TTTGGATGCC GAGGTCGTAG-3'

[0264] [Example 2-4. Evaluation of IMP production capacity of gcvPTH-enhanced, sdaA-deficient and glyA-enhanced integrated strains based on formate dehydrogenase-deficient and formate-dependent phosphoribosylamide formyltransferase-enhanced strains]

[0265] Flask titer evaluation was performed to measure the IMP production capacity of the strains prepared in Examples 2-3 and 2-4 and the parent strains. Corynebacterium stationis KCCM12151P, CJI-3188 and CJI-3189 were inoculated into a 14mL test tube containing less than 2.5mL of seed culture medium and incubated with shaking at 30°C and 170rpm for 24 hours. Then, 2mL of seed culture was inoculated into a 250mL corner baffle flask containing less than 29mL of production medium (24mL main culture medium + 5mL separate sterile culture medium) and incubated with shaking at 30°C and 170rpm for 72 hours. After the cultivation was completed, the IMP production capacity was measured by HPLC.

[0266] The composition of the seed culture medium and the fermentation medium is the same as that of Example 1-3.

[0267] The culture results of Corynebacterium stationis according to the combination of gcvPTH enhancement, glyA enhancement and sdaA deletion with formate dehydrogenase deletion and enhancement of formate-dependent phosphoribosylglycinamide formyltransferase are shown in Table 10 below.

[0268] [Table 10: Confirmation of IMP production by combination of gcvPTH enhancement, glyA enhancement, and sdaA deletion with formate dehydrogenase deletion and formate-dependent phosphoribosylglycinamide formyltransferase enhancement]

[0269] Strain number Import Type OD IMP(g / L) Concentration increase rate (%) KCCM12151P Control group 40.3 5.4 - CJI-3188 CJI-3163_Pn / gcvPTH 39.2 6.3 16.7 CJI-3189 CJI-3188_delsda::Pcj7 / glyA 39.3 6.8 25.9

[0270] It was confirmed that integration of fdh deletion, purT enhancement, and gcvPTH enhancement into the IMP-producing strain resulted in increased IMP production, and in addition, when sdaA deletion and glyA enhancement were integrated, the IMP production concentration was further increased.

[0271] [Example 3. Preparation of XMP-producing strain lacking formate dehydrogenase and enhanced formate-dependent phosphoribosylglycinamide formyltransferase and evaluation of XMP production capacity]

[0272] [Example 3-1. Preparation of formate dehydrogenase-deficient recombinant vector and XMP production strain]

[0273] An experiment was conducted to prepare a strain in which endogenous formate dehydrogenase was deleted in a Corynebacterium stationis strain producing XMP to confirm whether increasing formate by deleting formate dehydrogenase would lead to an increase in XMP production. Using the plasmid pDCM2 (Korean Patent Publication No. 10-2020-0136813) for deletion, insertion and replacement of genes in the chromosome of Corynebacterium, a vector for deleting the gene encoding formate dehydrogenase was prepared as follows.

[0274] PCR was performed using the gDNA (genomic DNA) of wild-type Corynebacterium stationis ATCC6872 as a template, using the primer pair of SEQ ID NO: 35 and 36 and the primer pair of SEQ ID NO: 37 and 38. PCR was performed under the following conditions: denaturation at 94°C for 5 min, followed by 30 cycles (denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute and 30 seconds), and then polymerization at 72°C for 5 min. The pDCM2 vector was treated with XbaI, and the above PCR products were subjected to fusion cloning. The fusion cloning was performed by HD cloning kit (Clontech) was used. The resulting plasmid was named pDCM2-del fdh.

[0275] The primer sequences used for vector preparation are as follows.

[0276]

Table 11

[0277] SEQ ID NO: name sequence 35 Primers 5'-ACCCGGGGAT CCTCTAGAGA CATGCTCAGC ACCTTTCTTC-3' 36 Primers 5'-AGCCTTAAGG CTACCGGATC-3' 37 Primers 5'-TCCGGTAGCCTTAAGGCTAT GCCGGATTTCGACGAGTATC-3' 38 Primers 5'-AAGCTTGCAT GCCTGCAGCG CCTTCAAGGA CGCCTAAG-3'

[0278] By electroporation, the above-mentioned vector is transformed into Corynebacterium stationis CJX1664 bacterial strain (KR 10-1950141B1), then in the selection medium containing 25mg / L kanamycin, the bacterial strain that the vector is inserted into chromosome is selected as a preliminary candidate bacterial strain. After this, in the bacterial strain where homologous recombination takes place, the bacterial strain of the gene deletion of the selection encoding formate dehydrogenase is selected using SEQ ID NO:35 and 38 primers. The selected bacterial strain is named as CJX1664_del fdh.

[0279] [Example 3-2. Preparation of formate-dependent phosphoribosylglycinamide formyltransferase-enhanced recombinant vector and XMP production strain]

[0280] An experiment to prepare a strain in which the expression of endogenous formate-dependent phosphoribosylglycinamide formyltransferase was enhanced in an XMP-producing Corynebacterium stationis strain was conducted to confirm whether the enhancement of formate-dependent phosphoribosylglycinamide formyltransferase would lead to an increase in XMP-producing ability.

[0281] Meanwhile, the expression of formate-dependent phosphoribosylglycinamide formyltransferase was enhanced in the formate dehydrogenase-deficient strain to confirm whether the increase in formate caused by formate dehydrogenase deficiency would further increase XMP production through formate-dependent phosphoribosylglycinamide formyltransferase.

[0282] The pDCM2-Pcj7 / purT vector prepared in Example 1-2, which replaced the promoter of the purT gene encoding formate ribose-dependent phosphoribosylamide formyltransferase with a stronger promoter, was transformed into Corynebacterium stationis CJX1664 strain (KR 10-1950141B1) and CJX1664_del fdh (a formate dehydrogenase deletion strain) by electroporation, and then the strain in which the vector was inserted into the chromosome was selected as a preliminary candidate strain in a selection medium containing 25 mg / L kanamycin. Thereafter, among the strains in which homologous recombination occurred, the primer pair of SEQ ID NO: 9 and 12 was used to select the strain in which the purT gene was connected to the Pcj promoter. The selected strains were named CJX1664_Pcj7 / purT and CJX1664_del fdh_Pcj7 / purT, respectively.

[0283] [Example 3-3. Evaluation of XMP production capacity of two strains: a formate dehydrogenase-deficient strain and a formate-dependent phosphoribosylglycinamide formyltransferase-enhanced strain]

[0284] The XMP production capabilities of the three strains prepared in Example 3-1 and Example 3-2 and the parent strain were evaluated by flask fermentation titer evaluation.

[0285] First, each strain was inoculated into a test tube with a diameter of 18 mm containing less than 2 mL of seed culture medium, and incubated with shaking at 30 ° C for 24 hours, and used as a seed culture solution. Then, 0.7 mL of seed culture solution was inoculated into a 250 mL corner baffle flask containing less than 32 mL of production culture medium (24 mL of main culture medium + 8 mL of sterile culture medium alone), and incubated for 75 hours at 30 ° C and 170 rpm. After the cultivation was completed, the XMP production capacity was measured by HPLC. The concentration and concentration increase rate of XMP in the culture solution of each test strain are shown in Table 3 below.

[0286]

Seed culture medium (pH 7.5)

[0287] Glucose 1%, peptone 1%, meat extract 1%, yeast extract 1%, NaCl 0.25%, adenine 100mg / L, guanine 100mg / L (based on 1L distilled water)

[0288]

XMP flask production medium (main medium)

[0289] Glucose 40g / L, magnesium sulfate 10g / L, calcium chloride 100mg / L, iron sulfate 20mg / L, manganese sulfate 10mg / L, zinc sulfate 10mg / L, copper sulfate 0.8mg / L, histidine 20mg / L, cystine 15mg / L, β-alanine 15mg / L, biotin 100μg / L, thiamine 5mg / L, adenine 50mg / L, guanine 25mg / L, niacin 15mg / L, pH 7.0

[0290]

XMP Flask Production Medium (Additional Sterile Medium)

[0291] Potassium dihydrogen phosphate 18g / L, potassium dihydrogen phosphate 42g / L, urea 7g / L, ammonium sulfate 5g / L (based on 1L distilled water)

[0292] The above experiment was repeated three times, and the average values ​​of the analysis results are shown in Table 12 below.

[0293] [Table 12: Comparison of XMP production capacity according to the deletion of formate dehydrogenase and the enhancement of formate-dependent phosphoribosylglycinamide formyltransferase]

[0294] strain XMP concentration (g / L) XMP concentration increase rate (%) CJX1664 4.27 - CJX1664_del fdh 4.40 3.04％ CJX1664_Pcj7 / purT 4.39 2.81％ CJX1664_del fdh-Pcj7 / purT 4.51 5.62％

[0295] As shown in Table 12 above, compared with the control CJX1664, the XMP production capacity of the CJX1664_del fdh strain and the CJX1664_Pcj7 / purT strain increased by 3.04% and 2.81%, respectively, and the XMP production capacity of CJX1664_del fdh-Pcj7 / purT increased by 5.62%. Based on the results, it was confirmed that increasing formate by deleting formate dehydrogenase can lead to increased XMP production, and when the formate-dependent phosphoribosylglycinamide formyltransferase with formate as a substrate is enhanced in combination, the XMP production capacity is further improved.

[0296] [Example 3-4. Confirmation of GMP production capacity of formate dehydrogenase-deficient strain and formate-dependent phosphoribosylglycinamide formyltransferase-enhanced strain]

[0297] The GMP production capability was confirmed in the following manner using the XMP culture fluids of the strains prepared in Example 3-1 and Example 302 and the control parent strain (see Korean Patent Publication No. 10-2011-0105662A).

[0298] Specifically, the strain was cultured using the fermentation titer evaluation method of Example 3-3. After the culture was completed, the production of XMP (5'-xanthine monophosphate) was measured using HPLC.

[0299] Thereafter, in order to convert the produced XMP into GMP, the following conversion additives and E. coli XMP ammonia enzyme were added to the flask fermentation solution, and then the conversion reaction was performed at 40° C. for 2.5 hours. As a result of the above experiment, Table 13 shows the conversion rate which takes into account the GMP production per consumed XMP.

[0300] [Table 13: Comparison of GMP production capacity according to the deletion of formate dehydrogenase and the enhancement of formate-dependent phosphoribosylglycinamide formyltransferase]

[0301]

[0302] As shown in Table 13 above, it was confirmed that GMP was produced by the conversion reaction of XMP produced by each strain.

[0303]

Conversion additives

[0304] Phytic acid 1.8g / L, MgSO4 4.8g / L, nymeen 3mL / L, xylene 2%, adenine 100mg / L, Na2HPO4 7.7g / L, glutamine 2g / L, glucose 46g / L

[0305] [Example 4. Preparation of gcvPTH-enhanced, sdaA-deficient and glyA-enhanced strains based on formate dehydrogenase-deficient and formate-dependent phosphoribosylamide formyltransferase-enhanced strains and evaluation of XMP production capacity]

[0306] [Example 4-1. Preparation of XMP-producing strain with additional enhancement of gcvPTH gene]

[0307] Based on the strain with fdh deletion and purT promoter enhancement, a strain was prepared in which a copy of the gcvPT gene was added in the form of the native promoter.

[0308] The pDCM2-Pn / gcvPTH vector prepared in Example 2-1 was transformed into the CJX1664_del fdh-Pcj7 / purT strain prepared in Example 3-2 by electroporation, and then the strain with the vector inserted on the chromosome was selected as a preliminary candidate strain in a selection medium containing 25 mg / L kanamycin. Thereafter, among the strains in which homologous recombination occurred, the strain to which the gcvPTH copy was added was selected using the primer pair of SEQ ID NO: 23 and 24. The selected strain was named CJI-CJX1664_delfdh-Pcj7 / purT-Pn / gcvPTH.

[0309] [Example 4-2. Preparation of XMP production strain with Sda deletion and glyA addition enhancement]

[0310] Based on the introduction of XMP production strains with purT enhancement and gcvPTH enhancement, in order to ensure a serine pool that can lead to glycine, a strain was prepared in which the endogenous gene sdaA of Corynebacterium stationis encoding the protein was deleted and the copy number of the glyA gene was added at the corresponding position to confirm the effect of further enhancing the glycine pool on XMP production.

[0311] The pDCM2-del_sdaA-Pn / glyA vector prepared in Example 2-3 was transformed into CJX1664_del fdh-Pcj7 / purT-Pn / gcvPTH prepared in Example 4-1 by electroporation, and then the strain with the vector inserted on the chromosome was selected as a preliminary candidate strain in a selection medium containing 25 mg / L kanamycin. Thereafter, among the strains in which homologous recombination occurred, the primer pair of SEQ ID NO: 34 and 35 was used to select the strain in which sdaA was deleted and the glyA gene connected to the Pcj7 promoter was inserted at the corresponding position. The strain obtained by the above method was named CJX1664_del fdh-Pcj7 / purT-Pn / gcvPTH_del sdaA-Pn / glyA.

[0312] [Example 4-3. Evaluation of XMP production capacity of gcvPTH-enhanced, sdaA-deficient and glyA-enhanced strains based on formate dehydrogenase-deficient and formate-dependent phosphoribosylamide formyltransferase-enhanced strains]

[0313] The strains prepared in Example 4-1 and Example 4-2 and the parent strain were evaluated for XMP production capacity by flask fermentation titer evaluation using the same method as Example 3-3. The above experiment was repeated three times, and the average values ​​of the analysis results are shown in Table 14 below.

[0314] [Table 14: Evaluation of XMP production capacity of sdaA-deficient, glyA-enhanced and gcvPTH-enhanced strains based on fdh-deficient and purT-enhanced strains]

[0315]

[0316] As shown in Table 14 above, it was confirmed that the XMP production capacity of CJX1664_del fdh_Pcj7 / purT_Pn / gcvPTH, in which the gcvPTH gene was further enhanced, was further enhanced compared to the CJX1664_del fdh_Pcj7 / purT strain, and the CJX1664_del fdh_Pcj7 / purT_Pn / gcvPTH_del sdaA_Pn / glyA strain, in which sdaA was further deleted and glyA was added, also showed an increase in XMP production. The results showed that when sdaA was deleted, glyA was enhanced, and gcvPTH was enhanced, fdh was deleted, purT was enhanced, and sdaA was deleted, and gcvPTH and glyA were enhanced together, the XMP production capacity was further enhanced.

[0317] [Example 4-4. Confirmation of GMP production capacity of gcvPTH-enhanced, sdaA-deficient and glyA-enhanced strains based on formate dehydrogenase-deficient and formate-dependent phosphoribosylglycinamide formyltransferase-enhanced strains]

[0318] Using the XMP culture fluids of the strains prepared in Example 4-1 and Example 4-2, GMP production was confirmed in the same manner as in Example 3-4. The results are shown in Table 15 below.

[0319] [Table 15: Confirmation of GMP production capacity of sdaA-deficient, glyA-enhanced and gcvPTH-enhanced strains based on fdh-deficient and purT-enhanced strains]

[0320]

[0321] As shown in Table 15 above, it was confirmed that GMP was produced by the conversion reaction of XMP produced by each strain.

[0322] In summary, those skilled in the art to which this application relates will be able to understand that this application may be embodied in other specific forms without modifying the technical concepts or basic features of this application. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of this application. On the contrary, this application is intended to cover not only exemplary embodiments, but also various alternatives, modifications, equivalents, and other embodiments that may be included in the purpose and scope of this application as defined in the appended claims.

[0323]

Claims

1. In a microorganism capable of producing purine nucleotides, the formate dehydrogenase activity is weakened compared with its endogenous activity.

2. The microorganism according to claim 1, wherein the formate dehydrogenase consists of any one or more amino acid sequences selected from SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and combinations thereof.

3. The microorganism according to claim 1, wherein the activity of any one or more proteins selected from the following (a) to (c) is further regulated: (a) the activity of glycine degrading enzyme is enhanced compared with its endogenous activity; (b) the serine hydroxymethyltransferase activity is enhanced compared to its endogenous activity; and (c) Formate-dependent phosphoribosylglycinamide formyltransferase activity is enhanced compared to its endogenous activity. The microorganism according to claim 1 , wherein the microorganism is a Corynebacterium microorganism. 5 . The microorganism according to claim 4 , wherein the Corynebacterium microorganism is Corynebacterium stationis .

6. The microorganism according to any one of claims 1 to 5, wherein the microorganism has an increased purine nucleotide production capacity compared to an unmodified microorganism.

7. A method for producing a purine nucleotide, comprising: A microorganism is cultured in a culture medium, wherein the formate dehydrogenase activity of the microorganism is attenuated compared to its endogenous activity.

8. The method according to claim 7, wherein the method further comprises recovering the target substance from the cultured microorganism, the culture product of the microorganism, the fermentation product of the microorganism or the culture medium.

9. A composition for producing purine nucleotides, comprising: Microorganisms, whose formate dehydrogenase activity is attenuated compared to its endogenous activity, a culture product of the microorganism, a fermentation product of the microorganism, or A combination of 2 or more of the above.

10. Use of a microorganism for producing purine nucleotides, wherein the formate dehydrogenase activity of the microorganism is attenuated compared to its endogenous activity.

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